A Method for Simultaneous Determination of 14 Carbonyl-Steroid Hormones in Human Serum by Ultra High Performance Liquid Chromatography–Tandem Mass Spectrometry

Abstract An improved method is described for the fast and sensitive determination of multiple endogenous steroid hormones in steroidogenic pathway by liquid chromatography–tandem mass spectrometry (LC–MS/MS), in which serum samples, after a simple protein precipitation and a derivatization by 2-hydrazinopyridine (HP), were subjected to LC–MS analysis. The protein precipitation of serum samples is achieved via adding acetonitrile, while the derivatization of endogenous steroid hormones in serum samples after protein precipitation is performed using 2-hydrazinopyridine as a derivatization reagent. The derivatives of the steroid hormones exhibit good detection sensitivity in LC–MS analysis. Linearity range of the improved method for the target steroid hormones is in two orders of magnitude and the correlation coefficients ( r ) are in the range from 0.9885 to 0.9998. The limits of detection (signal/noise = 3) range from 0.07 to 65.26 ng/mL. The recoveries are between 80.2 and 116.4% with acceptable intra- and inter-day relative standard deviations (RSDs) ranging from 0.3 to 18.9%. The improved method can be applied to explore the differences of steroid hormones between healthy controls and polycystic ovary syndrome patients. Graphic Abstract